Purification of NADH-cytochrome b5 reductase from sheep lung and its electrophoretic, spectral and some other properties.
1. NADH-cytochrome b5 reductase was purified from sheep lung microsomes in the presence of non-ionic and ionic detergents, Emulgen 913 and cholate, respectively. 2. The purification procedure involved the ion-exchange chromatography of the detergent solubilized microsomes on DEAE-cellulose. 3. Further purification and concentration of lung reductase was carried out with a second DEAE-cellulose column followed by the affinity column chromatography of partially purified reductase on 5'-ADP-agarose column. 4. The specific activity of sheep lung reductase was 638 mumol ferricyanide reduced/min/mg protein and the yield was 6% of the initial activity in microsomes. 5. The SDS-polyacrylamide gel electrophoresis of the purified lung reductase showed one protein band having the monomer mol. wt of 34,500 +/- 1500. In the presence of 0.4% deoxycholate, it existed as an active dimer having a mol. wt of 68,500. 6. Trypsin treated lung reductase showed two extra protein bands of mol. wts of 28,000 and 25,000 on 10% SDS-polyacrylamide gels. 7. The purified enzyme was found to contain FAD as prosthetic group and the absorption spectrum of lung reductase showed two peaks at 390 and 461 nm which were typical for flavoproteins and a shoulder at 490 nm. 8. The maximal activity of lung reductase was observed between pH 6.5-8.0 and at pH 6.8, when ferricyanide and partially purified sheep lung cytochrome b5 was used as electron acceptors, respectively.
Guray
T
T
Department of Biology, Middle East Technical University, Ankara, Turkey.
Arinç
E
E
D000818:Animals; D019826:Cholic Acid; D002793:Cholic Acids; D002846:Chromatography, Affinity; D003579:Cytochrome Reductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D042966:Cytochrome-B(5) Reductase; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005292:Ferricyanides / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008970:Molecular Weight; D012756:Sheep; D013053:Spectrophotometry; D014357:Trypsin / Q000494:pharmacology
D002793:Cholic Acids; D003902:Detergents; D005292:Ferricyanides; C007931:hexacyanoferrate III; D005182:Flavin-Adenine Dinucleotide; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D014357:Trypsin; D019826:Cholic Acid